Micro-arrays are tools for DNA and RNA molecular diagnostics. Detection of nucleic acids is possible using parallelisation techniques thereby enabling the investigation of several thousands of sequences in one reaction or experiment. Most applications focus on expression profiling for measuring adequately the expression of several thousands genes of interest. Detection of molecular binding on micro-arrays is visualized by use of special fluorescent dyes such as Cy3 and Cy5. Visualization of the binding is limited by the stability of the fluorescent marker, and in order to evaluate the processed micro-arrays, highly sophisticated and expensive laser scanning devices are required in addition to highly sophisticated software for analysing the data generated by laser scanning devices.
For the last thirty-five years, metal particles including gold and silver have been used as both contrast enhancement agents or light absorption labels in many different types of analytic and/or diagnostic applications. The great majority of these applications fall under the category of cytoimmunochemistry studies which have used gold or silver enhanced gold particles as markers to study structural aspects of cellular, subcellular, or tissue organization. In these studies, metal particles are usually detected and localized by electron microscopy, including scanning, transmission, and BEI (backscattered electron imaging). These methods take advantage of the electron dense nature of metals or the high atomic number of metals to facilitate the detection of the gold particles by virtue of the large numbers of secondary and backscattered electrons generated by the dense metal (see; Hayat, Immunogold-silver staining reference Page 1 and Chapters 1, 6-15; and Hayat, Colloid Gold reference Chapters 1, 5, 7 and others).
A number of patents describe the use of enzymatic methods or gold-based technology on micro-arrays to detect the presence of specific sequences of DNA. PCT patent application number WO 00/72018 (EP 1 179 180) (Advanced Array Technologies) describes the use of biotinylated DNA as probes against DNA samples immobilised on a glass micro-array, using gold (10 nm) labelled streptavidin as a visualisation agent.
US 2001/0010906A1 describes optimisation of capture probes design for sandwich hybridisation on solid carrier.
EP 1 164 201 describes the use of inverted detection for identifying and/or quantifying nucleotide target sequences on biochips using micro-fluidity techniques.
EP 1 096 024 describes a method for detection of homologue sequences after multiplex PCR for detecting Staphylococcus microorganisms.
AU8366001, AU7547501, CA2397280, WO0196604, AU736340, U.S. Pat. No. 6,214,560, CN1282378T, EP1023456, EP1021554, AU1294399, WO9920789 disclose a similar technique using gold labelled streptavidin particles of at least 80 nm for visualisation of bound nucleic acids on a glass micro-array using back scattered light.
U.S. Pat. No. 5,583,001, U.S. Pat. No. 196,306 and U.S. Pat. No. 5,731,158 disclose the use of in situ amplification techniques wherein the signal generated by the bound probe is amplified and visualised using enzymatic or gold based techniques. The catalysed amplification reporter deposit (CARD) technique was found to result in a signal amplification ranging from 10 to 100 fold and found to give equal results as the polymer based technique. The principle of CARD is widely used for several applications including electron microscopy, immunohistochemistry, ELISA, and in situ hybridization. The use of gold in a CARD based amplification has been described for the above mentioned techniques but real signal amplification on micro-arrays is not disclosed using gold or an enzymatic based technology.
U.S. Pat. No. 6,451,980 discloses a technique for signal enhancement of bi-specific antibody-polymer probe for use in immunoassay. Therein is described the use of bispecific antibodies where one part of the antibody complex recognises the antigen and the other part binds the polymer probe consisting of a poly-L-lysine backbone coated with “detectable signals”.
WO0206511 and AU8292001 (Genisphere) disclose an amplification technique applicable on micro-arrays, consisting of a dendrimer-based approach. Dendrimers are DNA molecules marked with a fluorescent dye. The special feature is that those molecules can form three dimensional structures by a type of hybridisation which results in a supermolecule heavily marked with Cy3 or Cy5 molecules. This leads to an enormous amplification of the signal generated by the bound nucleic acid of interest.
EP 1 230 396 and WO 01/36681 (Digene) disclose a technology detecting DNA/RNA hybrids on micro-arrays using a specific monoclonal antibody directed specifically to RNA/DNA hybrids with visualisation using fluorescent dyes.
WO 96/14314 disclose the use of a specific monoclonal antibody detecting DNA/PNA nucleic acid hybrids in solution and on a solid support.
For the detection of low concentrations of molecule components in the field of diagnostics, the methods of chemiluminescence and electrochemiluminescence are widely used. These methods provides a means to detect low concentrations of components by amplifying the number of luminescent molecules or photon generating events manyfold, the resulting “signal amplification” then allowing for detection of low concentration components. However, the above mentioned methods of signal amplification have associated limitations which makes the detection of components by these methods complicated, not easy to use, time consuming, and costly.
Problems of interference of chemical or enzymatic reactions, contamination, complicated and multi-step procedures, limited adaptability to single step “homogeneous” (non-separation) formats, and the requirement of costly and sophisticated instrumentation are areas that those in the art are constantly trying to improve.
Improvements has so far failed to provide means for the quantative and/or qualitative detection of molecules such as DNA, RNA, proteins, polypeptides without an evaluation step requiring an additional device such as a laser scanner, equipment to measure scattered light and/or specialised software. A low cost means of detection of components in samples is of importance in everyday fields of environmental science, veterinary medicine, pharmaceutical research, food and water quality control and the like. Furthermore a means which is simple to use, obviates the need for specialised training in equipment and/or protocols. Furthermore, the detection of substances at low concentrations (less than about 1 picomole substance/sample volume analyzed) is presently not possible without the use of fluorescent, luminescent, chemiluminescent, or electrochemiluminescent labels and other detection methods, all of which require optical reading devices to evaluate results.
It is a purpose of this invention not only to overcome the present day limitations, for example the disadvantages of light scattering-based diagnostic assays, but to also overcome the limitations and disadvantages of other non-light scattering methods such as signal amplification. This invention as described herein is easier to use, has greater detection sensitivity, and is capable of measuring components in samples across a wider concentration range than was previously possible. The present invention is broadly applicable to most sample types and assay formats as a signal generation and detection system for components.
The present invention provides a signal and detection system for the detection of components where the procedures can be simplified and the amount and types of steps and reagents reduced. The present invention provides for the quantitative and/or qualitative detection of single or multiple components in a sample. The present invention also provides for substantial reductions in the number of different tests and amounts of sample material that are analysed. Such reduction in the number of individual tests leads to reduced cost and waste production, especially medically-related waste that must be disposed of.